Calcium-dependent structural coupling between opposing globular domains of calmodulin involves the central helix

Area of Science:

• Biochemistry
• Structural Biology
• Spectroscopy

Background:

• Calmodulin (CaM) is a crucial calcium-binding protein that regulates numerous cellular processes.
• The central helix of CaM connects its globular domains and is involved in calcium binding.
• Understanding CaM's conformational dynamics is essential for elucidating its regulatory mechanisms.

Purpose of the Study:

• To investigate the structural dynamics and conformational heterogeneity of CaM's central helix.
• To determine how calcium binding affects the CaM central helix structure and domain arrangement.
• To explore the role of ionic strength and pH on CaM's conformational states.

Main Methods:

• Site-directed mutagenesis to create a double mutant (Tyr99Trp, Leu69Cys) in CaM.
• Fluorescence resonance energy transfer (FRET) using a Trp donor and an IAEDANS-labeled Cys acceptor.
• Spectroscopic analysis of CaM structure and conformational heterogeneity under varying conditions.

Main Results:

• Calcium binding induced a ~7 Å decrease in the distance between Trp99 and AEDANS-Cys69, indicating domain rearrangement.
• CaM's central helix showed minimal conformational heterogeneity change under physiological conditions (pH 7.5, 0.1 M KCl) upon calcium binding.
• Low ionic strength or pH altered CaM structure, decreasing central helix heterogeneity with calcium activation.

Conclusions:

• Calcium binding induces a conformational switch in CaM, altering the spatial arrangement of its globular domains.
• The central helix's conformational heterogeneity is largely independent of calcium under physiological conditions.
• Ionizable groups influencing the central helix structure play a role in CaM's rapid target protein activation.